The invention concerns an analytical element for the determination of an analyte containing in or on material which enables liquid transport between zones, a sample application zone and a detection zone located downstream thereof, wherein the detection zone contains a partner 1 of a specific binding pair 1 immobilized in such a manner that it is able to bind with partner 2 of the specific binding pair 1 which is not the analyte when this contacts it as well as a method for the determination of an analyte by means of specific binding pairs. The invention additionally concerns a kit for the determination of an analyte containing an analytical element.
Analytical elements are known from the prior art in which the reagents required to determine an analyte are present in or on carrier materials. Examples are: U.S. Pat. No. 4,861,711, U.S. Pat. No. 5,591,645 and EP-A-0 291 194. A common feature of the analytical elements described in these documents is that they are especially suitable for carrying out immunological detection methods. They comprise a sample application zone and a detection zone which is located downstream thereof. A liquid sample migrates through various zones between the sample application zone and detection zone as a result of capillary forces within a porous carrier material and thereby takes up the reagents that are necessary for detecting the analyte and reacts them with the analyte in the sample.
A binding partner is immobilized in the detection zone which is able to specifically bind the analyte to be determined. In the case of different analytes this requires that different binding partners for the analyte have to be immobilized on a solid phase.
It is also known from FIG. 1 of U.S. Pat. No. 4,861,711 in conjunction with the description, for example in column 5, line 57 to column 6 line 48 that a partner 1 of a specific binding pair 1 can be immobilized in the detection zone which does not bind the analyte but can be used universally because it binds an epitope as partner 2 of the specific binding pair 1 which is present on a substance which specifically binds the analyte. Hence the mobile complex of analyte and this binding substance is immobilized in the detection zone during the course of the detection reaction and is separated from non-complexed mobile reaction components.
A labelled substance that is specific for the analyte plays a very important role because only its binding to the analyte and the later immobilization of the complex formed composed of analyte and labelled substance directed against the analyte in the detection zone as well as the removal of the mobile non-reacted reaction components from the detection zone is able to indicate the presence of analyte in the liquid sample. The labelled substances known from the prior art are specific for the analyte i.e. in the case of sandwich assays they are labelled substances such as antibodies or antigens which react specifically with the analyte to be determined (antigen or antibody). However, this requires that depending on the analyte, different labelled specific binding partners for the analyte have to be prepared.
Numerous substances are known from the prior art for labelling. Whereas in the past radioactive labels with all their disadvantages were used, these labels were later replaced mainly by enzyme labels. Nowadays particulate labels, especially gold or latex particles, are mainly used in analytical elements as described in the previously described documents of the prior art. The preparation of a conjugate composed of a label and a substance binding specifically to the analyte is complicated and has to be optimized for each individual analyte-specific binding partner if it is intended to determine different analytes. In addition in analytical elements the material on which this conjugate is present and transported must be optimally adapted to the requirements from case to case. In this connection above all stability problems have often to be solved.
Therefore the object of the present invention was to provide a general purpose structure of an analytical element which can always be used independent of the analyte to be determined provided this analyte or a substance derived from the analyte and representing this analyte can be detected by specific pair binding.
This object is achieved by the subject matter in the invention as described in the claims.
The invention in particular concerns an analytical element for the determination of an analyte containing in or on material which-enables liquid transport between zones, a sample application zone and a detection zone located downstream thereof, wherein the detection zone contains a partner 1 of a specific binding pair 1 immobilized in such a manner that it is able to bind to partner 2 of the specific binding pair 1 which is not the analyte when it contacts it, characterized in that a labelled partner 1 of a specific binding pair 2 is present upstream of the detection zone impregnated on a material such that it can be detached by liquid and is able to bind to partner 2 of the specific binding pair 2 which is not the analyte when this contacts it, in which partner 2 of the specific binding pair 1 and partner 2 of the specific binding pair 2 are specifically bound to the analyte to be determined or by reaction involving the analyte to be determined are parts of a substance derived from and representing the analyte.
The invention also concerns a kit for the determination of an analyte containing an analytical element as characterized above as well as additionally containing at least one partner from the group of partner 2 of the specific binding pair 1 and partner 2 of the specific binding pair 2.
Finally the invention also concerns a method for the determination of an analyte by means of specific binding pairs characterized in that a substance derived from and representing the analyte which comprises partner 2 of a specific binding pair 1 and partner 2 of a specific binding pair 2 is contacted in an analytical element according to the invention for the determination of an analyte with a labelled partner of the specific binding pair 2, is moved by liquid transport in the analytical element towards the detection zone which is upstream of the sample application zone, is bound in the detection zone to partner 1 of the specific binding pair 1 and is determined on the basis of the label of partner 1 of the specific binding pair 2.
An essential feature of the analytical element according to the invention is that liquid can move within the analytical element towards the detection zone. Such a liquid flow is for example possible by gravitational force in an appropriately prepared hollow body. Devices which enable liquid transport by centrifugal force as a type of gravitational force are known for example from EP-B 0 052 769. However, analytical elements according to the invention preferably contain absorbent materials which are able to move liquid by capillary force. The materials of the individual zones of the analytical element according to the invention can in this connection be the same or different. It will often be the case that different zones are composed of different materials if these are to optimally fulfil their function.
Suitable potential absorbent capillary-active materials are basically all those which can be used to take up liquid in so-called dry tests as described for example in U.S. Pat. No. 4,861,711, U.S. Pat. No. 5,591,645 or EP-A-0 291 194. Porous materials such as membranes, for example nitrocellulose membranes have proven to be advantageous. However, it is also possible to use fibrous, absorbent matrix materials such as fleeces, fabrics or knitted fabrics. Fleeces are particularly preferred. Fibrous matrix materials can contain, glass, cellulose, cellulose derivatives, polyester, polyamide and also viscose, artificial wool and polyvinyl alcohol. Fleeces made of cellulose-based fibres, polymer fibres based on polyester and/or polyamide and an organic binding agent which has OH and/or ester groups as known from EP-B-0 326 135 can for example be used according to the invention. Fleece materials containing meltable copolyester fibres in addition to glass fibres, polyester fibres, polyamide fibres, cellulose fibres or cellulose derivative fibres as described in the European patent application 0 571 941 can also be used in the analytical element according to the invention. Papers such as tea bag paper are also suitable.
In order to improve the handling of the analytical element according to the invention, the absorbent capillary-active material or different absorbent capillary-active materials can be arranged on a stiff carrier material which is itself impermeable to liquid, does not negatively influence the liquid flow in the matrix material and is inert with regard to the reactions that occur on the analytical element. Polyester foil can be a preferred carrier material on which the matrix material enabling liquid transport is attached.
In the analytical element according to the invention the individual zones can be arranged on the carrier material on top of each other, next to one another or partially on top of and partially next to one another. An analytical element according to the invention is particularly preferred in which the sample application zone and detection zone are arranged next to one another on the carrier material. In this connection next to one another means that these zones are adjacent and in direct contact with one another or are arranged essentially in one plane separated by other zones.
xe2x80x9cIn accordance with one embodiment the analytical element of the present invention is provided with a sample application zone and a detection zone. The detection zone is located downstream of the application zone and the analytical element is designed to enable liquid transport between the two zones. In accordance with one embodiment, the analytical element comprises a material that promotes liquid transport between the application zone and the detection zone.xe2x80x9d
The sample application zone is the region of the analytical element according to the invention on which the sample is applied in which it is intended to determine whether a particular analyte or a substance derived from and representing this analyte is present and optionally in which amount it is present.
The detection zone is the region of the analytical element according to the invention in which it is determined whether the examined analyte or the substance derived from and representing the analyte was present in the sample applied to the analytical element. This determination can be qualitative, semiquantitative or quantitative. In this connection semiquantitative means that a specific concentration value is not determined for the analyte or for the substance derived from and representing the analyte but rather a concentration range is determined in which the analyte concentration is located.
xe2x80x9cAccording to one embodiment, the detection zone contains a partner 1 of-a specific binding pair 1 immobilized in such a manner that it is able to bind to a partner 2 of the specific binding pair 1 when partner 2 contacts partner 1, wherein said partner 2 is not the analyte. In addition the analytical element also includes a labelled partner 3 of a specific binding pair 2 present upstream of the detection zone. Labelled partner 3 is impregnated on a material such that it can be detached by liquid and is able to bind to a partner 4 of the specific binding pair 2 when partner 4 contacts partner 3, wherein said partner 4 is not the analyte.xe2x80x9d
xe2x80x9cIn one embodiment the specific binding pair 1 and specific binding pair 2 of the analytical element independently comprise a pair of specific binding partners selected from the group consisting of a hapten and an antibody, an antigen and an antibody, a lectin and a sugar/saccharide, a ligand and a receptor, avidin/streptavidin and biotin, a nucleic acid and a nucleic acid.xe2x80x9d
xe2x80x9cIn one embodiment, the labelled partner 3 is an antibody against partner 4, more particularly, labelled partner 3 is an antibody against digoxigenin or digoxin. In an alternative embodiment, labelled partner 3 is labelled with an enzyme or direct label. For example, the direct label can be a metal or latex particle. In these two embodiments, labelled partner 3 is typically located in the sample application zone.xe2x80x9d
Partner 1 of a specific binding pair 1 is immobilized in the detection zone in such a way that it is able to bind to partner 2 of the specific binding pair 1 which is not the analyte when this contacts it. The immobilization can be achieved by chemical reaction i.e. by formation of a covalent bond. However, it can also be achieved by adsorptive forces which includes all possibilities except for the formation of covalent bonds. A nitrocellulose membrane is frequently used for the detection zone to which proteins and also nucleic acids bind tightly when impregnated but without covalent binding.
According to the invention a labelled partner 1 of a specific binding pair 2 must also be in the analytical element in addition to partner 1 of a specific binding pair 1. This partner must hot be immobilized but must be present in an impregnated form that can be detached by liquid i.e. it must be possible to transport this labelled partner by liquid towards the detection zone. Advantageously it should be possible to completely i.e. quantitatively detach this labelled partner by as little liquid as possible from the matrix material on which it is impregnated. Fleeces have proven to be particularly suitable as a matrix material for this as described for example in EP-B-0 326 135.
Specific binding pairs are known from the prior art and include for example pairs such as hapten and antibody, antigen and antibody, lectin and sugar or saccharide, avidin or streptavidin and biotin as well as nucleic acid and nucleic acid, ligand and receptor. In this connection an antigen can be any molecule against which one can experimentally produce antibodies. An antigen can also be an antibody or a particular site of an antibody which is referred to as an epitope and is specifically recognized and bound by an antibody. Nucleic acids should be understood as all possible forms of nucleic acids which are able to bind via complementary bases. DNA, RNA and also nucleic acid analogues such as peptide nucleic acids (PNA see for example WO 92/20702) are specifically mentioned but are not a definitive list. Ligand and receptor quite generally refer to a specific binding interaction between two partners such as between a hormone and hormone receptor.